Method for increasing plasma volume by administering a plasma expander comprising basic alpha keratose

ABSTRACT

A liquid plasma expander or resuscitation fluid composition for use in a subject in need thereof, comprising, consisting of; or consisting essentially of: (a) a keratin derivative (preferably alpha keratose, gamma keratose, or combinations thereof, and with basic alpha keratose preferred over acidic alpha keratose); and (b) an electrolyte solution, with the keratin derivative solubilized in the electrolyte solution to form a homogeneous liquid composition. Blood substitutes formed therefrom and methods of making and using the same are also described.

RELATED APPLICATIONS

This application claims priority to and is a divisional of U.S. patentapplication Ser. No. 11/205,800, filed Aug. 17, 2005 now U.S. Pat. No.7,439,012, and also claims the benefit of U.S. provisional patentapplication Ser. No. 60/602,207, filed Aug. 17, 2004, the disclosure ofeach of which is incorporated by reference herein in its entirety.

GOVERNMENT FUNDING

This invention was made with Government support under contract numberW81XWH-04-1-0105 from the United States Army. The US Government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention is generally related to plasma expanders and bloodsubstitutes, and is particularly related to high viscosity plasmaexpanders and blood substitutes.

BACKGROUND OF THE INVENTION

Many biocompatible polymeric materials have been investigated aspotential plasma expanders and/or blood substitutes. Historically, therehave been two approaches: 1) the use of synthetic compounds that arebiocompatible, and 2) the use of biological materials that arepolymeric. In the first category, materials such as hydroxyethyl starchand perfluorocarbon liquid have been evaluated (See, e.g., S. Kasper etal., J. Clin. Anesth. 13, 486-90 (2001); T. Kaneki et al., Resuscitation52, 101-08 (2002); R. Spence et al., Art Cells, Blood Sibst., ImmobilBiotech. 22, 955-63 (1999)). In the second group is gelatin, albumin,and crosslinlked hemoglobin (I. Tigchelaar et al., Eur. J. Cardo-thor.Surg. 11, 626-32 (1997); S. Gould et al., World J. Surg. 20, 1200-7(1996). More recently, alpha-keratose has been suggested. See A. Widra,U.S. Pat. No. 6,746,836 (Jun. 8, 2004).

Because the functional consequences of changing the flow properties ofblood are not readily predictable, the development of plasma expandersand/or blood substitutes is a complicated matter. In arterial bloodvessels (diameter >100 micron) blood viscosity is proportional tohematocrit (Hct) squared, and in the smaller vessels it is linearlyproportional to Hct. In the systemic circulation, Hct is approximatelyconstant down to 100 micron diameter vessels. It falls monotonicallydown to the capillaries where it is approximately half of the systemicvalue. The reverse occurs in the venous circulation, where it is higherthan arterial because of fluid filtration in the microcirculation.

In acute conditions such as accompanying severe trauma, the decrease ofHct is not deemed dangerous until the transfusion trigger (bloodhemoglobin content beyond which a blood transfusion is indicated) isreached. However, this exposes the vasculature to low blood viscositywhen conventional plasma expanders are used to maintain blood volume.There appears to be no well-defined benefit to lowering blood viscosity,excepting when it is pathologically high, and lowering blood viscositythrough hemodilution is considered to have no adverse effects.Richardson and Guyton determined that changes in blood viscosity areaccompanied by compensatory changes in cardiac output, which compensatefor changes in intrinsic oxygen carrying capacity of blood due tochanges in Hct (T. Richardson et al., Am. J. Physiol. 197, 1167-70(1959)). This was confirmed systemically and in the microcirculation (K.Messmer, Surg. Clins. N. Am. 55, 659-78 (1975); S. Mirhashemi et al.,Am. J. Physiol 254 (Heart Circ. Physiol. 13) H411-16 (1988); A. Tsai etal., Int. J. Microcirc: Clin. Exp 10, 317-34 (1991)). Empirically, thetransfusion trigger is set at 7 g Hb/dl (Hct˜22%).

Microvascular Hcts are lower than systemic due to the presence of aplasma layer that proportionally occupies a greater portion of thevessel lumen, thus blood viscosity is also lower. The transition frommacro to microcirculation in terms of vessel dimensions. Hct, andhemodynamics is gradual. Blood theological properties also changegradually and blood viscosity in the circulation depends on location.The reduction of Hct with a crystalloid or colloidal plasma expandertends to equalize the theological properties of blood and viscositythroughout the circulation.

When a plasma expander is used to remedy hemorrhage, systemic Hctdecreases, significantly reducing blood viscosity in large vessels dueto the squared dependence of viscosity on Hct. Viscosity of blood insmall vessels is much less affected since Hct is low to begin with.Conversely, small vessel blood viscosity is greatly influenced by theviscosity of the plasma expander. If its viscosity is low, bloodviscosity drops significantly in the small vessels as well as in thelarge vessels, although for somewhat different reasons. In conventionaltheory, this reduction in viscosity increases blood flow and may improveoxygen delivery.

However, the literature supports the concept that high viscosity plasmais either beneficial, or has no adverse effect in conditions of extremehemodilution. Waschke et al. found that cerebral perfusion is notchanged when blood is replaced with fluids of the same intrinsic oxygencarrying capacity over a range of viscosities varying from 1.4 cp to 7.7cp (K. Waschke et al., J. Cereb. Blood Flow & Metab. 14, 871-976 (1994))Krieter et al., varied the viscosity of plasma by adding dextran 500 kDaltons (Da) and found that medians in tissue pO₂ in skeletal musclewhere maximal at a plasma viscosity of 3 cp, while for liver the maximumoccurred at 2 cp (H. Krieter et al., Acta Anaest. Scad. 39, 326-44(1995)). In general they found that up to a 3 fold increase in bloodplasma viscosity had no effect on tissue oxygenation and organ perfusionwhen blood was hemodiluted. de Witt et al., found elevation of plasmaviscosity causes sustained NO-mediated dilatation in the hamster musclemicrocirculation (C. deWitt et al., Pflugers Arch. 434, 54-61 (1997)).

Hct reductions should improve blood perfusion through the increase ofblood fluidity. However at a Hct near to and beyond the transfusiontrigger the heart cannot further increase flow and as viscosity falls,so does blood pressure. The fall of pressure is deleterious for tissueperfusion because it decreases functional capillary density (FCD) in thenormal circulation and in hypotension following hemorrhage (L. Lindbomet al., Int. J. Microcirc: Clin. Exp 4, 121-7 (1985)). FCD is a criticalmicrovascular parameter in survival during acute blood losses. In ahamster model subjected to 4-hr 40 mmHg hemorrhagic shock, the fall ofFCD accurately predicts outcome and separates survivors from nonsurvivors when this parameter decreases below 40% of control (H. Kergeret al., Am. J. Physiol 270 (Heart. Circ. Physiol. 39), H827-36 (1996)).

High viscosity plasma restores mean arterial pressure (MAP) inhypotension without vasoconstriction. Furthermore, the shift of pressureand pressure gradients from the systemic to the peripheral circulationincreases blood flow, which in combination with increased plasmaviscosity maintains shear stress in the microcirculation. This is neededfor shear stress dependant NO and prostaglandin release from theendothelium and to maintain FCD (J. Frangos et al., Science 227, 1477-79(1985)). Conversely, reduced blood viscosity decreases shear stress andthe release of vasodilators, causing vasoconstriction and offsetting anybenefit of reducing the theological component of vascular resistance.Since resistance depends on the 4^(th) power of vascular radius and the1^(st) power of blood viscosity, the effect of reducing blood viscositywith a low viscosity plasma expander is that it reduces oxygen deliveryto the tissue once blood viscosity falls below a threshold value. Thisthreshold has been determined in our experimental model as about 2.5 cp.

Tissue perfusion with reduced blood viscosity may be deleterious at thecellular/endothelial level. There is evidence that genes are activatedfollowing changes in the mechanical environment of cells. It is alsobeen established that the endothelium uniquely responds to changes inits mechanical and oxygen environment according to programmed geneticschemes. Among these responses is the mechanism for apoptosis (cell selfdestruction), which is activated through a genetically controlledsuicide process that eliminates cells no longer needed or excessivelydamaged. In this context, hemodilution with low viscosity plasmaexpanders may cause cellular and tissue damage due to hypoxia and/or tothe reduced vessel wall shear stress. Hypoxia/ischemia may contribute toendothelial impairment due to inflammatory reactions. Activation ofendothelium, platelets and neutrophils, leading to additional damagethrough the liberation of cytokines, can induce endothelial apoptosis(B. Robaye et al., Am. J. Pathol. 38, 447-53 (1991)).

Studies in a hamster model show that extreme hemodilution (where Hct is20% of control) with dextran 70 kDa, causes hypotension and a drop inFCD to near pathological values (A. Tsai et al., Proc. Natl. Acacd. Sci.USA 95, 6590-5 (1998); A. Tsai. Transfusion 41, 1290-8 (2001)). This isprevented by increasing plasma viscosity so that the diluted blood has asystemic viscosity of about 2.8 cp, which was achieved by infusingdextran 500 kDa. Thus, high viscosity plasma substitutes can be analternative to the use of blood for maintaining MAP and an adequatelevel of FCD (A. Tsai et al., Biorheology 38, 229-37 (2001)). However,the known high viscosity plasma expanders such as gelatin, albumin,hydroxyethyl starch, polyvinyl-pyrrolidine, and dextran are all eithernon-human derived or synthetic. As such, each suffers from considerablelimitations in their clinical applicability due to biocompatibility,cost, or both. What is needed is a fluid based on a substantiallybiocompatible material that is inexpensive, pathogen free and ambientstorable. Resuscitation fluids based on keratins offer this potential.

Human hair is one of the few autologous tissues that can be obtainedwithout additional surgery. It is also a rich source of keratins.Equally important, the biocompatibility of keratins within a species,and indeed across species is high, making allogenous and xenogenouskeratins viable candidates for medical applications. The keratins foundin hair, wool, and other keratinous tissues can be extracted andpurified using methods known in the art, and used for formulating plasmasubstitutes with fluid properties that will maintain MAP and FCD.Depending on the species from whence the keratins come, thebiocompatibility can also be optimized with human hair keratins beingthe most optimal. Keratin fluids are inexpensive to produce, can besterilized, and are stable under ambient temperature storage.

However, the keratin-based fluid described in A. Widra would not appearto be the most optimized resuscitation medium based on the new paradigmof preserving FCD for three important reasons. First, the type ofkeratin used in the experiments was a highly hydrolyzed form ofkeratose, represented in Scheme 1 below, which is not likely to becapable of attaining the viscosic properties required by theapplication.

Second, hydrolyzed forms of keratose are compatible with blood in thatthey do not instigate appreciable levels of red blood cell aggregation,but their oncotic pressure is too low to be of benefit. Third, lesshydrolyzed, high molecular weight forms of keratose tend to aggregatered blood cells, thus making the material deleterious to the restorationof FCD. Hence their remains a need for new approaches to developingkeratin-based high viscosity plasma substitutes.

SUMMARY OF THE INVENTION

A first aspect of the invention is a liquid plasma expander orresuscitation fluid composition for use in a subject in need thereof,comprising, consisting of, or consisting essentially of: (a) a keratinderivative (preferably alpha keratose, gamma keratose, or combinationsthereof); and (b) an electrolyte solution, with the keratin derivativesolubilized in the electrolyte solution to form a homogeneous liquidcomposition.

A particular aspect of the foregoing is a liquid plasma expandercomposition in which the keratin derivative comprises alpha keratose,where the alpha keratose consists of at least 80, 90, 95 or 99 percentby weight of basic alpha keratose (or more), and where the alphakeratose consists of not more than 20, 10, 5 or 1 percent by weight ofacidic alpha keratose (or less).

A particular aspect of the foregoing is a liquid plasma expandercomposition for use in a subject in need thereof, comprising, consistingof or consisting essentially of: (a) from 0.1 to 10 or 20 percent byweight of basic alpha keratose; (b) from 0 to 5 or 10 percent by weightof gamma keratose; and (c) from 80 or 90 to 99.9 percent by weight of anelectrolyte solution. Preferably, the basic alpha keratose and the gammakeratose are solubilized in said electrolyte solution to form ahomogeneous liquid composition. Preferably the homogeneous liquidcomposition has a pH of 7 to 8 or 9; preferably the homogeneous liquidcomposition has an osmolarity of 100 or 200 to 500 or 600milliosmoles/Liter; and preferably the homogeneous liquid compositionhas a viscosity of 2 or 4 to 15 or 20 centipoise. Preferably thehomogeneous liquid composition, when contacted to red blood cells, formsaggregates of said blood cells of less than 25 microns in diameter.

The basic alpha keratose is preferably produced by separating basicalpha keratose from a mixture of acidic and basic alpha keratose, e.g.,by ion exchange chromatography, and preferably the basic alpha keratosehas an average molecular weight of from 10 to 100 or 200 kilodaltons.Optionally but preferably the process further comprises the steps ofre-disolving said basic alpha-keratose in a denaturing solution (such asa buffer solution), optionally in the presence of a chelating agent tocomplex trace metals, and the re-precipitating the basic alpha keratosefrom the denaturing solution. It will be appreciated that thecomposition preferably contains not more than 5, 2, 1, or 0.1 percent byweight of acidic alpha keratose, or less.

A second aspect of the present invention is a blood substitutecomposition for use in a subject in need thereof, comprising, consistingof, or consisting essentially of: a plasma expander or resuscitationfluid as described above, and red blood cells (RBCs) where the RBCs forman essentially single cell suspension of RBCs therein.

A third aspect of the present invention is a method of increasing plasmavolume in a subject in need thereof, comprising administering thesubject a plasma expander or resuscitation fluid composition asdescribed above in an amount effective to increase the plasma volume ofsaid subject.

A fourth aspect of the present invention is a method of increasing thevolume of available blood substitute for treatment in a subject in needthereof, comprising the steps of: (a) obtaining a volume of donor bloodand determining the Hct; (b) separating and isolating the RBCs from saiddonated blood; and (c) diluting said isolated RBCs to a final Hct of notless than 10% of the original Hct but not greater than 70% by adding anappropriate amount of the plasma expander composition as describedabove.

A further aspect of the present invention is the use of a keratinderivative as described herein for the preparation of a plasma expanderor resuscitation fluid for carrying out a method as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the osmolarity of various keratin resuscitation fluids.Diamonds represent alpha-keratose fluids; squares represent gammakeratose fluids; triangles represent alpha-kerateine fluids; circlesrepresent gamma-kerateine fluids.

FIG. 2 a-d shows the thrombogenic potential of keratin resuscitationfluids as evaluated by whole blood aggregometry.

FIG. 2 a shows whole blood aggregation with 1 mg/mL Collagen.

FIG. 2 b shows whole blood aggregation with 2% alpha-keratose.

FIG. 2 c shows whole blood aggregation with 2% gamma keratose.

FIG. 2 d shows whole blood aggregation with 2% alpha-kerateine.

FIG. 3. (A, C) Oxidation or (B, D) reduction, followed by extraction inaqueous base removes essentially all the cortical proteins from hairfibers.

FIG. 4. Viscosity of keratin solutions at 37° C. Improved solubility andhigh viscosity was achieved with α-kerateine using a derivatizationprocedure that produced an scarboxymethyl analog. The α-keratose samplewas obtained using a recently developed low-hydrolysis extractionmethod.

FIG. 5. Viscosity (a) and osmolarity (b) curves for keratose fluidsformulated in Ringer's lactate solution and adjusted to pH 7.4. Thetarget viscosity for a hyperviscous fluid is approximately 8 cP (wholeblood is ca. 4, depending on location in the body). The osmolarity ofwhole blood is approximately 300 mOsm/kg. These data were obtained usingan early extraction protocol that resulted in excessive hydrolysis, assuggested by the relatively low viscosity achieved at concentrations ashigh as 10 weight %.

FIG. 6. Impedance measurement of lightly citrated human whole blood withthe addition of 100 μL of γ-keratose (a), α-keratose (b), and collagen 1(c; +control). Lack of a detectable baseline shift suggestsnon-thrombogenic properties and good blood compatibility.

FIG. 7. Lightly citrated samples of 4% γ-keratose (a) and α-keratose (b)in Ringer's lactate mixed 1:1 with whole blood. These initial keratosefluids were of low viscosity and consequently did not instigate RBCaggregation or platelet activation. SDS-PAGE analysis confirmed that theproteins were excessively hydrolyzed which may have contributed to theirapparent blood compatibility.

FIG. 8. Fluids prepared in PBS at pH 7.4 with 5 weight % acidic (a) andbasic (b) α-keratose were mixed 1:1 with whole blood. RBC aggregation isevident in the acidic fluid as positively charged keratin proteinsinteract with the blood cells. The negatively charged basic keratinsrepel the negatively charged blood cells and prevent aggregation.

FIG. 9. Histology of major organs harvested from rats subjected tosimultaneous fluid exchange. All animals recovered from the surgerieswithout incident and no deleterious effects were noted in any of thetissues. (Magnification ×200)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“Solubilized” as used herein refers to a compound that is carried by asolvent to form a homogeneous mixture therewith, without precipitationor separation of that compound from the solvent. Examples of suchhomogeneous mixtures include solutions, suspensions, dispersions, andmicroemulsions. Preferably the homogeneous mixtures do not scattervisible light (i.e., are “clear” on visual inspection).

“Dissolved” as used herein refers to a compound or solute carried byanother liquid or solvent in the form of a single-phase solution.

“Subjects” (or “patients”) to be treated with the methods andcompositions described herein include both human subjects and animalsubjects (particularly other mammalian subjects such as dogs, cats,horses, monkeys, etc.) for veterinary purposes. Human subjects areparticularly preferred. The subjects may be male or female and may beany age, including neonate, infant, juvenile, adolescent, adult, andgeriatric subjects.

“Plasma expander” as used herein refers to a composition that may beused to increase the volume of blood plasma in a subject in needthereof. The plasma expander itself may be blood-free. In someembodiments the plasma expander may serve as a resuscitation fluid.

“Resuscitation fluid” as used herein refers to a plasma expander thatfurther serves to maintain, pretent further decrease, and/or preventaccelerated decrease of functional capillary density (FCD) in a subject.Plasma expanders of the present invention are preferably alsoresuscitation fluids.

“Keratin derivative” as used herein refers to any keratin derivative ormixture thereof, including but not limited to alpha keratose, gammakeratose, alpha kerateine, gamma kerateine, meta-keratin, keratinintermediate filaments, and combinations thereof.

“Electrolyte solution” as used herein includes saline solution, mixedsalt or buffer solutions such as Ringers solution, Lactated Ringerssolution, and combinations thereof.

“Blood substitute” as used herein refers to any fluid capable ofreplicating the biochemical (e.g. oxygen carrying) and biomechanical(e.g. viscosic) capabilities of whole blood.

The disclosures of all United States patents cited herein are to beincorporated by reference herein in their entirety.

1. Keratin materials. Keratin materials are derived from any suitablesource including but not limited to wool and human hair. In oneembodiment keratin is derived from end-cut human hair, obtained frombarbershops and salons. The material is washed in hot water and milddetergent, dried, and extracted with a nonpolar organic solvent(typically hexane or ether) to remove residual oil prior to use.

Keratose Fractions. Keratose fractions are obtained by any suitabletechnique. In one embodiment they are obtained using the method ofAlexander and coworkers (P. Alexander et al., Biochem. J. 46, 27-32(1950)). Basically, the hair is reacted with an aqueous solution ofperacetic acid at concentrations of less than ten percent at roomtemperature for 24 hours. The solution is filtered and thealpha-keratose fraction precipitated by addition of mineral acid to a pHof ca. 4. The alpha-keratose is separated by filtration, washed withadditional acid, followed by dehydration with alcohol, and then driedunder vacuum. Increased purity can be achieved by redissolving thekeratose in a denaturing solution such as 7M urea, aqueous ammoniumhydroxide solution, or 20 mM tris base buffer solution,re-precipitating, re-dissolving, dialyzing against deionized water, andre-precipitating at pH 4.

The gamma-keratose fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and driedunder vacuum. Increased purity can be achieved by redissolving thekeratose in a denaturing solution such as 7M urea, aqueous ammoniumhydroxide solution, or 20 mM tris buffer solution, reducing the pH to 4by addition of a mineral acid, removing any solids that form,neutralizing the supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and reprecipitating byaddition to alcohol. The amount of alcohol consumed in these steps canbe minimized by first concentrating the keratose solution bydistillation.

Kerateine Fractions. Kerateine fractions are obtained using acombination of the methods of Bradbury and Chapman (J. Bradbury et al.,Aust. J. Biol. Sci. 17, 960-72 (1964)) and Goddard and Michaelis (D.Goddard et al., J. Biol. Chem. 106, 605-14 (1934)). Essentially, thecuticle of the hair fibers is removed ultrasonically in order to avoidexcessive hydrolysis and allow efficient reduction of cortical disulfidebonds in a second step. The hair is placed in a solution ofdichloroacetic acid and subjected to treatment with an ultrasonic probe.Further refinements of this method indicate that conditions using 80%dichloroacetic acid, solid to liquid of 1:16, and an ultrasonic power of180 Watts are optimal (H. Ando et al., Sen'i Gakkaishi 31(3), T81-85(1975)). Solid fragments are removed from solution by filtration, rinsedand air dried, followed by sieving to isolate the hair fibers fromremoved cuticle cells.

Following ultrasonic removal of the cuticle, alpha- and gamma-kerateinesare obtained by reaction of the denuded fibers with mercaptoethanol.Specifically, a low hydrolysis method will be used at acidic pH (E.Thompson et al., Aust. J. Biol. Sci. 15, 757-68 (1962)). In a typicalreaction, hair is extracted for 24 hours with 4M mercaptoethanol thathas been adjusted to pH 5 by additional of a small amount of potassiumhydroxide in deoxygenated water containing 0.02M acetate buffer and0.001M surfactant.

The solution is filtered and the alpha-kerateine fraction precipitatedby addition of mineral acid to a pH of ca. 4. The alpha-kerateine isseparated by filtration, washed with additional acid, followed bydehydration with alcohol, and then dried under vacuum. Increased purityis achieved by re-dissolving the kerateine in a denaturing solution suchas 7M urea, aqueous ammonium hydroxide solution, or 20 mM tris buffersolution, re-precipitating, re-dissolving, dialyzing against deionizedwater, and re-precipitating at pH 4.

The gamma-kerateine fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and driedunder vacuum. Increased purity is achieved by redissolving the kerateinein a denaturing solution such as 7M urea, aqueous ammonium hydroxidesolution, or 20 mM tris buffer solution, reducing the pH to 4 byaddition of a mineral acid, removing any solids that form, neutralizingthe supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and reprecipitating byaddition to alcohol. The amount of alcohol consumed in these steps canbe minimized by first concentrating the keratose solution bydistillation.

In an alternate method, the kerateine fractions are obtained by reactingthe hair with an aqueous solution of sodium thioglycolate.

Meta-Keratins. Meta-keratins are synthesized from both the alpha- andgamma-fractions of kerateine using substantially the same procedures.Basically, the kerateine is dissolved in a denaturing solution such as7M urea, aqueous ammonium hydroxide solution, or 20 mM tris buffersolution. Pure oxygen is bubbled through the solution to initiateoxidative coupling reactions of cysteine groups. The progress of thereaction is monitored by an increase in molecular weight as measuredusing SDS-PAGE. Oxygen is continually bubbled through the reactionsolution until a doubling or tripling of molecular weight is achieved.The pH of the denaturing solution can be adjusted to neutrality to avoidhydrolysis of the proteins by addition of mineral acid.

Keratin Intermediate Filaments. IFs of human hair fibers are obtainedusing the method of Thomas and coworkers (H. Thomas et al., Int. J.Biol. Macromol. 8, 258-64 (1986)). This is essentially a chemicaletching method that reacts away the keratin matrix that serves to “glue”the IFs in place, thereby leaving the IFs behind. In a typicalextraction process, swelling of the cuticle and sulfitolysis of matrixproteins is achieved using 0.2M Na₂SO₃, 0.1M Na₂O₆S₄ in 8M urea and 0.1MTris-HCl buffer at pH 9. The extraction proceeds at room temperature for24 hours. After concentrating, the dissolved matrix keratins and IFs areprecipitated by addition of zinc acetate solution to a pH of ca. 6. TheIFs are then separated from the matrix keratins by dialysis against0.05M tetraborate solution. Increased purity is obtained byprecipitating the dialyzed solution with zinc acetate, redissolving theIFs in sodium citrate, dialyzing against distilled water, and thenfreeze drying the sample.

2. Formulations. Dry powders may be formed of keratin derivatives asdescribed above in accordance with known techniques such as freezedrying or lyophilization. In some embodiments, a liquid plasma expandercomposition of the invention may be produced by mixing such a dry powdercomposition form with an aqueous solution to produce a homogeneousliquid plasma expander composition comprising an electrolyte solutionhaving said keratin derivative solubilized therein. The mixing step canbe carried out at any suitable temperature, typically room temperature,and can be carried out by any suitable technique such as stirring,shaking, agitation, etc. The salts and other constituent ingredients ofthe electrolyte solution (e.g., all ingredients except the keratinderivative and the water) may be contained entirely in the dry powder,entirely within the aqueous composition, or may be distributed betweenthe dry powder and the aqueous composition. For example, in someembodiments, at least a portion of the constituents of the electrolytesolution are contained in the dry powder.

In the composition the keratin derivatives (particularly alpha and/orgamma kerateine and alpha and/or gamma keratoses) have an averagemolecular weight of from about 10 to 70 or 100 kiloDaltons. Otherkeratin derivatives, particularly meta-keratins, may have higher averagemolecular weights, e.g., up to 200 or 300 kiloDaltons. In general, thekeratin derivative (this term including combinations of derivatives) maybe included in the composition in an amount of from about 0.1, 0.5 or 1percent by weight up to 3, 4, 5, or 10 percent by weight. Thecomposition when mixed preferably has a viscosity of about 1 or 1.5 to4, 8, 10 or 20 centipoise. Viscosity at any concentration can bemodulated by changing the ratio of alpha to gamma keratose. Studies haveshown that viscosities as high as 7.7 have no deleterious effects (seeK. Waschke et al., J. Cereb. Blood Flow & Metab. 14, 871-976 (1994) andH. Krieter et al., Acta Anaest. Scad. 39, 326-44 (1995)).

The electrolyte solution may be any suitable electrolyte solution,including but not limited to saline solution (particularly normalsaline), Ringer's solution, lactated Ringer's solution, commerciallyavailable solutions such as NORMOSOL®-R isotonic fluid (available fromAbbott Laboratories, Chicago, Ill. USA), and combinations thereof.Examples of suitable electrolyte compositions include but are notlimited to those described in U.S. Pat. No. 6,746,836 to Widra. Thecomplete composition when mixed preferably has an osmolarity of 200 to400 milliosmoles/Liter and a pH of about 7 to 8.

The composition is preferably sterile and non-pryogenic. The compositionmay be provided preformed and aspectically packaged in a suitablecontainer, such as a flexible polymeric bag or bottle, or may beprovided as a kit of sterile dry powder in one container and sterileaqueous solution in a separate container for mixing just prior to use.When provided pre-formed and packaged in a sterile container thecomposition preferably has a shelf life of at least 4 or 6 months (up to2 or 3 years or more) at room temperature, prior to substantial loss ofviscosity (e.g., more than 10 or 20 percent) and/or substantialprecipitation of the keratin derivative (e.g., settling detectable uponvisual inspection).

3. Subjects and administration. As noted above, the present inventionprovides a method of increasing plasma volume in a subject (human oranimal) in need thereof, or of replacing or augmenting whole blood in asubject in need thereof, comprising administering said subject a plasmaexpander or blood substitute, respectively, as described above in anamount effective to resuscitate said subject.

The dose or volume administered to the subject will depend upon factorssuch as the age and general health of the subject, the particularcondition of the subject, the disorder being treated and the severity ofthat disorder, etc., and can be determined by skilled persons inaccordance with known techniques. In some embodiments the volumeadministered will be between about 0.5 or 1 to 50 or 70 mL per Kgsubject body weight. In some embodiments the volume administered will befrom 0.1 or 0.2 Liters up to 3 or 4 Liters total per subject. Forexample, the resuscitation fluid can be utilized up to the transfusiontrigger (Hct. of ca. 22%). This means that for an average man with a Hctof ca. 54% and a blood volume of ca. 5 L, he can be transfused with upto 3 L of plasma expander/resuscitation fluid of the invention beforeblood transfusion is necessary. Hct and blood volume vary by gender andage, so target volumes would vary accordingly. In another example, forneonatal shock, the composition may be administered in aliquots ofbetween 2 to 10 mL per Kg every few minutes as necessary.

In general, subjects in need thereof include subjects in shock,typically due to acute or chronic bleeding. Particular subjects includebut are not limited to subjects suffering from a laceration, incision orother injury to any portion of the body, such as an extremity, orsuffering from internal injury to an organ such as the liver. Theinternal injury may be acute, as resulting from trauma, or chronic, suchas a bleeding ulcer or diverticulitis.

Subjects that may be treated by the methods of the invention includepatients undergoing a surgical procedure, where bleeding is aconsequence of the surgical procedure. In cases where the surgicalprocedure is elective, the subject may donate whole blood before thesurgery to be used in the preparation of an autologous keratin-basedblood substitute.

Subjects that may be treated by the methods of the invention includeburn victims, particularly severe burn victims, where blood fluid isrequired to carry and clear burn toxins from the body to avoid organpoisoning and organ failure.

In addition to administer to subjects, the plasma expander compositionsof the present invention may be used as bath, storage or rinsecompositions for organs (particularly mammalian organs of the samespecies as described above in connection with subjects) in connectionwith organ transplant procedures. Suitable organs include but are notlimited to kidney, heart, lung, liver, etc.

EXAMPLE 1 Production of Keratoses

50 grams of clean dried hair are oxidized using 1,000 mL of 2weight/volume % peracetic acid by gentle shaking at 37° C. for 12 hours.The oxidized hair is recovered by sieve and rinsed free of residualoxidant using copious amounts of deionized (DI) water. The damp hair isextracted with 2,000 mL of 0.1M tris base at 37° C. with gentle shakingfor 3 hours. The hair is separated by sieve and the liquid retained. Theswollen hair is further extracted with 2,000 mL of DI water at 37° C.with gentle shaking for 1 hour. The hair is separated by sieve and theliquid retained. A third and final extraction of the swollen hair isaccomplished with 25000 mL of DI water at 37° C. with gentle shaking for1 hour. The hair is separated by sieve and the liquid retained. Thethree extracts are combined and residual solids removed bycentrifugation and filtration. The keratose solution is titrated to pH 6by dropwise addition of acid and loaded onto a preconditioned ionicexchange column containing a weak ion exchange resin such as DEALSepharose™. The column is washed with an equal volume of 10 mM trisbuffer at pH 6 and the eluent saved for another use. The bound keratoseis removed from the column by washing with 100 mM tris buffer with 2MNaCl at pH 12. This fraction is referred to as basic keratose. The basickeratose is further separated into its gamma and alpha fractions byselective precipitation. For example, basic alpha-keratose is isolatedby reducing the pH of the solution to 4.2 with dropwise addition ofacid. The precipitate that forms is isolated by centrifugation and/orfiltration. The subject resuscitation fluid is formulated using eitherpure alpha or a mixture of alpha and gamma keratoses. Regardless, thebasic keratose solution is neutralized by addition of acid and dialyzedby tangential flow ultrafiltration against DI water for approximately 12hours (nominal low molecular weight cutoff of 5 kDa). The dialyzedsample is concentrated by removal on excess water on a rotary evaporatorand the basic keratose powder isolated by lyophilization. Resuscitationfluids are formulated from this powder by addition to phosphate-bufferedsaline at pH 7.4 and a concentration sufficient to achieve a viscosityof between 4 and 20 centipoise, preferably 8 centipoise.

EXAMPLE 2 Production of Alpha-Keratose Fraction

Alpha-keratose is isolated from the extract solution prepared in Example1 by dropwise addition of acid until the pH of the solution reachesapproximately 4.2. Preferred acids include sulfuric, hydrochloric, andacetic acid. A most preferred acid is concentrated hydrochloric acid.Precipitation of the alpha fraction begins at around pH 6.0 andcontinues until approximately 4.2. Fractional precipitation can beutilized to isolate different ranges of protein with differentisoelectric properties. Solid alpha-keratose is recovered bycentrifugation or filtration. The alpha-keratose is further purified byre-dissolving the solids in a denaturing solution. The same denaturingsolutions as those utilized for extraction can be used, however apreferred denaturing solution is Trizma base (also called tris base).Ethylene diamine tetraacetic acid (EDTA) is added to complex tracemetals formed in hair. A preferred denaturing solution is 20 mM trisbase with 20 mM EDTA. The alpha-keratose is re-precipitated from thissolution by dropwise addition of hydrochloric acid to a final pH of 4.2.Isolation of the solid is by centrifugation or filtration. This processcan be repeated several times to further purify the alpha-keratose.

EXAMPLE 3 Production of Gamma-Keratose Fraction

The gamma-keratose fraction is isolated by precipitation to awater-miscible non-solvent. Suitable non-solvents include ethanol,methanol, acetone, and the like. A most preferred non-solvent isethanol. To affect precipitation, the gamma-keratose solution can beconcentrated by evaporation of excess water. After removal of thealpha-keratose, the concentration of gamma-keratose from a typicalextraction solution is approximately 1-2%. This solution can beconcentrated to approximately 10-20% by removal of 90% of the water.This can be done using vacuum distillation. After concentration, thegamma-keratose solution is added dropwise to an excess of coldnon-solvent. A most preferred method is to concentrate thegamma-keratose solution to approximately 10% and add it dropwise to an8-fold excess of cold ethanol. The gamma-keratose is isolated bycentrifugation or filtration and dried. Suitable methods for dryinginclude freeze drying (lyophilization), air drying, vacuum drying, orspray drying. A most preferred method is freeze drying.

EXAMPLE 4 Production of Kerateines

A preferred method for the production of kerateines is by reduction withthioglycolic acid or beta-mercaptoethanol. A most preferred reductant isthioglycolic acid. Preferred concentrations range from 1 to 10 molar(M), the most preferred being approximately 1.0M. Those skilled in theart will recognize that slight modifications to the concentration can bemade to affect varying degrees of reduction, with concomitantalterations in pH, reaction time, temperature, and liquid to solidratio. A preferred pH is between 9 and 11. A most preferred pH is 10.2.The pH of the reduction solution is altered by addition of base.Preferred bases include transition metal hydroxides and ammoniumhydroxide. A most preferred base is sodium hydroxide. The pH adjustmentis affected by dropwise addition of a saturated solution of sodiumhydroxide in water to the reductant solution. A preferred reductiontemperature is between 0 and 100 degrees Celsius. A most preferredreduction temperature is 37° C. A preferred reduction time is between0.5 and 24 hours. A most preferred reduction time is 12 hours. Apreferred liquid to solid ratio is from 5 to 100:1. A most preferredratio is 20:1.

EXAMPLE 5 Production of Alpha and Gamma-Kerateine Fractions

Once the hair is reduced, the procedures for extracting and isolatingthe kerateines are identical as those described for the alpha- andgamma-keratoses. The only additional consideration is the relativesolubility of kerateines. The relative solubility rankings in water isgamma-keratose>alpha-keratose>gamma-kerateine>alpha-kerateine from mostto least soluble. That being the case, the production of resuscitationfluids from kerateines is facilitated by re-dissolving precipitatedsolutions in 20 mM tris base with 20 mM EDTA and dialyzing them. Typicaldialysis conditions are 1 to 2% solution of kerateines dialyzed againstdeionized water for 24 to 72 hours. Those skilled in the art willrecognize that other methods exist for the removal of low molecularweight contaminants in addition to dialysis (e.g. microfiltration,chromatography, and the like). The use of tris base is only required forinitial solubilization of the kerateines. Once dissolved, the kerateinesare stable in solution without the denaturing agent. Therefore, thedenaturing agent can be removed without the resultant precipitation ofkerateines. The final concentration of kerateines in these purifiedsolutions can be adjusted by the addition/removal of water.

EXAMPLE 6 Formulation of Plasma Expander

Regardless of the form of the keratin, a final plasma expandercomposition is, in one embodiment, preferably formulated in Ringer'slactate. This electrolyte solution has the following formulation for1,000 mL:

3.1 g sodium lactate

6.0 g sodium chloride

300 mg potassium chloride

200 mg calcium chloride

The Ringer's lactate/keratin solution is formulated to match the desiredviscosic and oncotic properties of blood. Suitable viscosities rangefrom 1.0 to 5.0 centipoise. A most preferred viscosity is 2.5centipoise. Suitable osmolarity ranges are from 100 to 500 mOsm/L. Amost preferred osmolarity is approximately 300 mOsm. Alternatively,phosphate-buffered saline can also be used.

EXAMPLE 7 Preparation of Alpha-Keratose

Human hair was obtained from a local barber shop, cut in to piecesapproximately ½ inch in length, washed with mild detergent and warmwater, dried in air, washed with ethanol, and dried in air. 10 grams ofthis clean, degreased, dry hair was oxidized in 200 mL of 8weight/volume (w/v) % of peracetic acid at 4° C. for 24 hours. The hairand oxidation solution was placed in a 2 L polyethylene jar and shakenon a reciprocating table shaker at 100 rpm. After oxidation, the liquidwas removed by sieve and the hair rinsed with a copious amount ofdeionized (DI) water. The oxidized hair was extracted with 500 mL of a0.3M sodium hydroxide solution at 4° C. for 24 hours. The extraction wasperformed in a 2 L polyethylene jar and shaken on a reciprocating tableshaker at 100 rpm. After extraction, residual cuticle was removed bysieve and the liquid recovered. Small particulates were removed bycentrifugation at 6,000 rpm for 30 minutes. The alpha-keratose wasprecipitated from the resulting liquid by dropwise addition ofconcentrated HCl to a pH of 4.2. The alpha-keratose was recovered bycentrifugation at 2,000 rpm for 20 minutes and the remaining liquid(“supernatant”) retained for future use. The alpha-keratose wasre-dissolved in 20 mM tris base with 20 mM EDTA, re-precipitated bydropwise addition of HCl to a pH of 4.2. The alpha-keratose wasrecovered by centrifugation at 2,000 rpm for 20 minutes, re-dissolved in20 mM tris base with 20 mM EDTA, and dialyzed against DI water usingdialysis tubing with a low molecular weight cutoff (LMWCO) of 14,200.

EXAMPLE 8 Preparation of Gamma-Keratose

The supernatant from the extraction in Example 7 (i.e. pH 4.2 solutionfrom the first precipitation of alpha-keratose) was concentrated 10-foldon a rotary evaporator using siphon vacuum and a water bath temperatureof 50° C. The gamma-keratose was precipitated from this viscous solutionby dropwise addition to an 8-fold excess of cold ethanol. Thegamma-keratose was recovered by centrifugation at 2,000 rpm for 20minutes, re-dissolved in a minimum volume of 20 mM tris base with 20 mMEDTA and residual alpha-keratose removed by acidification to pH 4.2 andremoval of any precipitate that formed. The gamma-keratose wasre-precipitated by dropwise addition of the remaining supernatant to an8-fold excess of cold ethanol, and recovered by centrifugation at 2,000rpm for 20 minutes. The gamma-keratose was dissolved in 20 mM tris basewith 20 mM EDTA and dialyzed against DI water using dialysis tubing witha LMWCO of 3,500.

EXAMPLE 9 Preparation of Alpha-Kerateine

Human hair was obtained from a local barber shop, cut in to piecesapproximately ½ inch in length, washed with mild detergent and warmwater, dried in air, washed with ethanol, and dried in air. 10 grams ofthis clean, degreased, dry hair was reduced in 200 mL of 1M sodiumthioglycolate at 4° C. for 24 hours. The sodium thioglycolate solutionhad been prepared by mixing thioglycolic acid with water, adjusting thepH to 10.2 by addition of a saturated solution of sodium hydroxide, anddiluting to a final thioglycolic acid concentration of 1 mole per liter.The hair and reduction solution was placed in a 2 L polyethylene jar andshaken on a reciprocating table shaker at 100 rpm. After reduction, theliquid was removed by sieve and the hair rinsed with a copious amount ofdeionized (DI) water. The reduced hair was extracted with 500 mL of a0.3M sodium hydroxide solution at 4° C. for 24 hours. The extraction wasperformed in a 2 L polyethylene jar and shaken on a reciprocating tableshaker at 100 rpm. After extraction, residual cuticle was removed bysieve and the liquid recovered. Small particulates were removed bycentrifugation at 6,000 rpm for 30 minutes. The alpha-kerateine wasprecipitated from the resulting liquid by dropwise addition ofconcentrated HCl to a pH of 4.2. The alpha-kerateine was recovered bycentrifugation at 2,000 rpm for 20 minutes and the supernatant retainedfor future use. The alpha-kerateine was re-dissolved in 20 mM tris basewith 20 mM EDTA, re-precipitated by dropwise addition of HCl to a pH of4.2. The alpha-kerateine was recovered by centrifugation at 2,000 rpmfor 20 minutes, re-dissolved in 20 mM tris base with 20 mM EDTA, anddialyzed against DI water using dialysis tubing with a low molecularweight cutoff (LMWCO) of 14,200.

EXAMPLE 10 Preparation of Gamma-Kerateine

The supernatant from the extraction in Example 9 (i.e. pH 4.2 solutionfrom the first precipitation of alpha-kerateine) was concentrated10-fold on a rotary evaporator using siphon vacuum and a water bathtemperature of 50° C. The gamma-kerateine was precipitated from thisviscous solution by dropwise addition to an 8-fold excess of coldethanol. The gamma-kerateine was recovered by centrifugation at 2,000rpm for 20 minutes, re-dissolved in a minimum volume of 20 mM tris basewith 20 mM EDTA, and residual alpha-keratose removed by acidification topH 4.2 and removal of any precipitate that formed. The gamma-keratosewas re-precipitated by dropwise addition of the remaining supernatant toan 8-fold excess of cold ethanol, and recovered by centrifugation at2,000 rpm for 20 minutes. The gamma-kerateine was dissolved in 20 mMtris base with 20 mM EDTA and dialyzed against DI water using dialysistubing with a LMWCO of 3,500.

EXAMPLE 11 Preparation of Plasma Expanders

A solution of Ringer's lactate was prepared according to the followingformula:

-   -   3.1 g sodium lactate    -   6.0 g sodium chloride    -   300 mg potassium chloride    -   200 mg calcium chloride    -   Dilute to 1,000 mL with DI water        Solutions of alpha- and gamma-keratose, and alpha- and        gamma-kerateine were prepared at concentrations of 0.5, 1.0,        2.0, 3.0, and 4.0 weight percent in Ringer's lactate. The        osmolarity of each solution was measured on an Osmette A model        5002 osmometer (Precision Systems, Inc., Natick, Mass.). The        data set forth in FIG. 1 was obtained.

EXAMPLE 12 Properties of Keratin Plasma Expanders

The keratin resuscitation fluids from example 11 were evaluated forthrombogenic potential by whole blood aggregometry. Fresh whole bloodwas drawn from healthy human volunteers and citrated by addition of 1 mLof 0.5 w/v % of trisodium citrate to 9 mL of whole blood. For eachsample analysis, 750 μL of citrated blood was mixed 1:1 with Ringer'slactate solution and warmed to 37° C. in a cuvette placed in the testwell of a Whole Blood Aggregometer, model 591 (Chrono-Log Corporation,Havertown, Pa.). The instrument was coupled to a PowerLab® 8sp analog todigital converter and computer running Chart 5 v5.1 data acquisitionsoftware (AD Instruments, Colorado Springs, Colo.). An impedance probewas placed in the cuvette and the resistance of the blood solutionmeasured over time. After at least 2 minutes of steady baselinemeasurement, 100 μL of the test solution was injected into the bloodsolution. Any change in resistance was measured on the impedance probe.An increase in signal would be indicative of platelet aggregation andpotential thrombogenicity. Keratin solutions were compared to a negativecontrol, Ringer's lactate, and a positive control, 1 mg/mL collagen Isolution. Data for the positive control and 2% alpha-keratose,gamma-keratose, and alpha-kerateine solutions are shown in FIG. 2.

EXAMPLE 13 Further Studies and Optimization

The extraction method development detailed in Examples 1 to 12 abovefocused on the production of keratoses and kerateines. The protocol forobtaining kerateines from hair fibers made use of a reductant thatconverts cystine crosslinks to cysteine residues. The initial reductionstep, coupled with subsequent extractions with tris base, was highlyeffective at removing the cortical proteins from human hair fibers (FIG.3). However, the thiol-functional cysteines are reactive and it was soondiscovered that once the keratin is precipitated, some of the materialwill not readily re-dissolve. In fact, even in storage at −80° C., thekerateine samples, particularly the α-kerateines, would becomecompletely insoluble even at basic pH. These samples became sointractable over time that it was not even possible to reduce them asecond time in order to completely dissolve the proteins.

In an attempt to resolve this solubility problem, we utilized aderivatization procedure discussed by Crewther et al. (The Chemistry ofKeratins. Anfinsen C B Jr., Anson M L, Edsall J T, and Richards F M,Editors. Advances in protein chemistry 1965. Academic Press. New York:191-346). Briefly, once the kerateines had been extracted from the hairand before precipitation of the alpha fraction, an s-carboxymethyl groupcan be grafted onto the cysteine residue by reaction with iodoaceticacid. This was accomplished by dialyzing the crude extract solution toremove residual reductant, then adding an excess of iodoacetic acid tothe protein solution. After reaction for several hours, the alpha andgamma s-carboxymethylkerateines (SCMK) could be separated as usual. Thismethod was successful in producing α-SCMK that was soluble at neutralpH, and of relatively high viscosity (FIG. 4). However, upon testingwith whole blood, the sample was determined to instigate excessive RBCaggregation. Although no further testing of the α-SCMK was conductedafter it was determined that RBCs aggregate in the presence of thismaterial, recent developments discussed later in this section suggestthat α-SCMK can be further purified to remove the causative material.

In contrast to the kerateine materials, and as expected, keratosesamples demonstrated no apparent solubility problems. This is due to theconversion of cystine to non-reactive and hydrophilic sulfonic acidresidues during the oxidative reaction rather than the highly reactivecysteine residues created during the reduction protocol. Preparation ofviscous solutions was relatively simple and fluids formulated inRinger's lactate were prepared and tested for viscosity and osmolarity(FIG. 5). The resulting viscosity of the α-keratose sample wasunacceptably low, even at a relatively high concentration of 10 weightpercent. The viscosity of the γ-keratose was also too low to beconsidered effective in the FCD model of resuscitation, but this wasexpected since the difference in molecular weight between alpha andgamma is nearly 5-fold. The osmolarity obtained from these samples waswithin acceptable limits (FIG. 5 b)

RBC aggregation studies of these two fluids were conducted using a wholeblood aggregometer. A technique was developed wherein human blood waslightly citrated so as to provide reasonable working time withoutinterfering with platelet function. Results of these experiments for thekeratose solutions at 4 weight percent and a positive control, collagen1, are shown in FIG. 6. This technique measures a change in impedance asRBC and platelets aggregate on a microprobe immersed in a 1:1 mixture ofblood and DI water. After baseline equilibration, a small amount of thetest solution is added and the change in impedance across the sensor ismonitored. As can be seen in FIG. 6, there is essentially no resultingbaseline deflection when the keratose fluids are added, suggesting thatthe RBC are not aggregated and the platelets similarly are not activatedby this material. In contrast, collagen 1, a known thrombogenic protein,elicits an immediate baseline shift. Further evidence of bloodcompatibility was obtained from microscopic examination of thesemixtures. Light micrographs show no visible evidence of RBC aggregationor platelet activation in slightly citrated samples (FIG. 7).

To further demonstrate the biocompatibility and safety of these keratosefluids, a 4 weight percent solution of α-keratose in Ringer's lactatewas prepared and sterile filtered. This solution was used in a top loadstudy in mice. The objective of these experiments was simply to assessthe safety of our fluid formulation in a conservative model. A smallbolus representing 10% of the total blood volume of the mouse was usedto assess the systemic effects of a small amount of keratin biomaterialin the circulation. All animals recovered without incident. Chemistryand CBC analysis showed that the animals had recovered a normal bloodprofile within the 24 hour period (Tables 1 and 2, respectively).

TABLE 1 Blood chemistry results for samples taken from mice subjected toa 10% top load IC injection (Control = no injection; RL = Ringer'slactate; keratin = 4 w/v % α-kertose) Glucose ALT Alb Alk P AST Bili-TBUN Ca²⁺ Creat Phos TP (mg/dL) (U/L) (g/dL) (U/L) (U/L) (mg/dL) (mg/dL)(mg/dL) (mg/dL) (mg/dL) (g/dL) Day 1 Control 157 41 2.8 17 54 0.8 20 9.90.30 6.2 4.7 RL 228 47 2.9 5 80 1.5 17 9.9 0.27 7.4 5.3 Keratin 155 382.6 7 44 0.7 20 9.7 0.17 7.2 4.9 Day 5 Control 270 32 2.9 9 54 0.9 187.2 0.22 6.3 5.3 RL 203 26 2.6 31 71 0.6 19 7.3 0.25 6.9 4.6 Keratin 21421 2.8 6 53 0.5 22 7.3 0.18 6.0 5.0

TABLE 2 Electrolytes and CBC from blood taken from mice subjected to a10% top load IC injection (Control = no injection; RL = Ringer'slactate; keratin = 4 w/v % α-kertose) WBC RBC NA* K⁺ Cl⁻ PCV (%) Hgb(g/dL) MCV (fl) (×1000) (×1000) (mmol/L) (mmol/L) (mmol/L) Day 1 Control28 13 46.6 41 6.01 144 5.2 111 RL 32 14.4 47.4 54 6.69 146 5.4 110Keratin 27 12.3 49 23 5.59 147 5.5 111 Day 5 Control 41 16.6 51.4 15.27.97 142 8.7 106 RL 43 16.9 49.7 5.5 8.74 143 7.4 111 Keratin 35 16 47.71.9* 7.32 144 8.1 113 *Sample viscosity was to high to produceconsistent results

It was apparent from these data that although RBC aggregation propertiesand cardiovascular compatibility were favorable, viscosity of thesesamples was too low to be effective in the FCD model. In hindsight, itwas probably due in part to the excessive hydrolysis, particularly ofthe α-keratose, that the fluids did not instigate RBC aggregation andwere highly compatible in the circulation. Sodiumdodecylsulfate-poly(acrylamide) gel electrophoresis (SDSPAGE) analysisof these samples confirmed that hydrolysis was prevalent (data notshown). A new extraction protocol was developed wherein the initialoxidation step was unchanged, but the tris base concentration in theinitial extraction step was halved. In addition, second and thirdextraction steps were added using DI water that served not only todilute residual tris base, but also increased the yield of protein (seeExample 1 above).

Viscosity of the α-keratose fraction isolated using this method was wellwithin acceptable limits (FIG. 4). However, a fluid formulation in PBSstill demonstrated excessive RBC aggregation characteristics. To addressthis, a simple ion exchange purification step was devised. A sample oflow-hydrolysis α-keratose was produced and dissolved at ca. 2 weight %in 100 mM tris base and the pH adjusted to 8.5 by addition of HCl. It iswell published in the literature that hair keratin proteins exist asacidic and basic families. The terms “acidic” and “basic” aredescriptors commonly used in the literature to describe sub-families ofkeratins that have different isoelectric points. Both sub-classes aresoluble at neutral pH and can be effectively separatedchromatographically. Ion exchange chromatography is a convenient,inexpensive, and scalable method for separating such mixtures.

Samples of acidic and basic α-keratose were generated using a flashchromatography column containing a commercially available resin. Thefractions were dialyzed against DI water, concentrated, and freezedried. Resuscitation fluid formulations using these fractions wereprepared at 5 weight percent concentration and their RBC aggregationcharacteristics evaluated with fresh whole human blood. As is shown inFIG. 8, the ion exchange chromatography was highly effective andseparating the aggregation phenomenon. Basic α-keratose was essentiallyfree from interactions with blood cells while the acidic α-keratosecaused excessive aggregation.

Based on these results confirming the isolation of a viscous, bloodcompatible keratin biomaterial, a resuscitation fluid formulation wastested in a simultaneous fluid exchange model. Again, the goal of thisexperiment was to assess the safety of the keratin fluid, albeit in amore aggressive model than previously described. Relatively large malerats were catheterized so that fluid could be simultaneous exchangedusing two synchronized syringe pumps. Large volumes of blood wereexchanged with PBS (control) and basic α-keratose fluid as a means totest the interaction of the keratin biomaterial in a dynamic circulatorysystem. All of the rats recovered from the procedure without incident(Table 3) and were sacrificed after 24 hours. Histology on the vitalorgans showed no deleterious effects with PBS or keratin (FIG. 9).

TABLE 3 Results of CBC and blood chemistry from samples taken 24 hoursafter simultaneous fluid exchange in male Fisher rats PBS Keratin Test25% 50% 75% 50% PCV (%) 24.4 18.7 15.7 20.2 Hgb (g/dl) 9.6 8.3 7.1 8.9MCV (fl) 54 53.3 54 54.2 WBC (×1000) 4.7 9.6 10 12.8 RBC (×1000) 4.523.51 2.91 3.73 Glucose 157 141 109 96 (mg/dl) ALT (U/l) 76 76 143 148Alb (g/dl) 3.6 3.1 3.1 1.6 Alk P (U/l) 87 130 121 131 AST (U/l) 378 152339 472 Bili-T (mg/dl) 0.5 0.6 0.9 0.9 BUN (mg/dl) 14 10 14 14 Ca 2+(mg/dl) 7.7 8.4 8.5 8.2 Creat (mg/dl) 0.51 0.48 0.42 0.59 Phos (mg/dl) 87.9 8.2 7.1 TP (g/dl) 6.1 5.9 5.6 6.1Materials and Methods

Animal testing was performed under an approved protocol. Regulations ofthe Wake Forest University Health Sciences Animal Care and Use Committeewere followed at all times.

Keratose Fractions. Keratose fractions were obtained using the method ofAlexander and coworkers (Alexander P, Hudson R F, and Fox M. Thereaction of oxidizing agents with wool: The division of cysteine intotwo fractions of widely differing reactivities. Biochem J 1950;46:27-32) and processed as described in Example 1 above.

Viscosity Measurements. Sample solutions were prepared at keratinconcentrations ranging from 1 to 10 weight percent. The viscosity ofeach solution was tested dynamically on a Viscometer (Model DV-I+,Brookfield Engineering Laboratories Inc.), using a cone and plategeometry with a cone angle of 0.02 radians. The samples were maintainedat 37° C. using a constant frequency of 30 rotations per minute. Thefollowing analysis procedure was followed: 1) stabilize the testgeometry at 37° C.; 2) open the heated chamber and load sample; and 3)close the chamber and allow ca. 2 minutes for the temperature to reach37° C. before the measurement was initiated.

Osmolality Measurements Osmometry was conducted on an Osmette A model5002 osmometer (Precision Systems, Inc., Natick, Mass.). Solutions atkeratin concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 weight percentwere evaluated by adding 2 mL in a cuvette and measuring freezing pointdepression.

Whole Blood Aggregation Measurements. Aggregometry experiments wereperformed using a Whole Blood Aggregometer, model 591 (Chrono-LogCorporation, Havertown, Pa.), coupled to a PowerLab®, 8sp analog todigital converter and PC running Chart 5 v5.1 data acquisition software(AD Instruments, Colorado Springs, Colo.). Additionally, 1:1 mixtures of4 wt. % keratin solutions with whole blood were observed by opticalmicroscopy for evidence of RBC aggregation.

Mouse Top Load Study. CD-1 outbred mice were anesthetized with 3%isoflurane and immobilized on a procedure table. The heart was locatedvisually and by palpation and a small 27.5 gauge needle inserted intothe heart; a small amount of blood was withdrawn to ensure the locationof the needle. A bolus of 4 weight % keratin that represented andestimated 10% of the total blood volume of the mouse was slowlyinjected. Ringer's lactate was used as a control. After the needle waswithdrawn, the animal was allowed to recover. After 24 hours and againafter 5 days, ca. 1 mL of blood was drawn using a similar procedure forCBC and chemistry.

Ion Exchange Chromatography. Samples were loaded on to a 200 mL flashchromatography column containing DEAE Sepharose™ weak anionic ionexchange resin in 10 mM tris base at pH 6. Basic α-keratose was elutedfrom the column with 100 mM tris base plus 2M NaCl at pH 12. The eluatewas neutralized and dialyzed against DI water using an ultrafiltrationtangential flow cartridge with a nominal low molecular weight cutoff of5 KDa, concentrated by rotary evaporation, and freeze dried.

Rat Fluid Exchange Study Under general anesthesia, subcutaneousincisions were made in the neck and thigh of male Fisher rats weighing300 to 350 grams. Angiocatheters connected to two syringe pumps wereinserted into the lumen of the femoral and jugular veins so thatsimultaneous infusion and withdraw could be performed. The syringe pumpswere synchronized to deliver and withdraw at the same rate (ca. 1.0mL/min). The fluid exchange transpired over approximately 15 to 20minutes, depending on animal weight, during which the animal's vitalsigns were monitored. Fluids included a phosphate buffered salinecontrol and basic α-keratose. After fluid exchange, the catheters wereremoved and hemostasis confirmed prior to wound closure in 2 layers withabsorbable sutures. Approximately 24 hours after recovery, the animalswere sacrificed and blood and vital organs harvested for analysis.

Blood Chemistry. Arterial pO₂, pCO₂ and pH at 37° C. using a pH/bloodgas analyzer (Chiron Diagnostics, Model 248) were measured from arterialblood sampled from the carotid artery into heparizined capillary tubes.Hemoglobin content of blood was determined from a drop of blood using ahandheld photometer device (B-Hemoglobin Photometer Hemocue, Angelholm,Sweden). Whole blood lactate was measured from a 25 μL sample using aYSI Sport Lactate Analyzer.

Tissue Histology. Vital organs were removed and flash frozen in liquidnitrogen. The tissues were microtomed, fixed, and stained withhematoxylin and eosin (H&E).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of increasing plasma volume in a subject in need thereof,comprising administering said subject a liquid plasma expander in anamount effective to increase the plasma volume of said subject, whereinsaid plasma expander consists essentially of: (a) from 0.1 to 10 percentby weight of basic alpha keratose; said basic alpha keratose produced bythe process of separating basic alpha keratose from a mixture of acidicand basic alpha keratose by ion exchange chromatography; and with saidbasic alpha keratose having an average molecular weight of from 10 to100 kilodaltons; (b) from 0 to 5 percent by weight of gamma keratose;and (c) from 90 to 99.9 percent by weight of an electrolyte solution;with said basic alpha keratose and said gamma keratose solubilized insaid electrolyte solution to form a homogeneous liquid compositionhaving: (i) a pH of 7-8; (ii) an osmolarity of 200 to 500milliosmoles/Liter; and (iii) a viscosity of 2 to 20 centipoise at atemperature of 37 degrees Celsius as determined with a Brookfieldviscometer having a cone and plate geometry with a cone angle of 0.02radians at a constant frequency of 30 rotations per minute.
 2. Themethod of claim 1, wherein said subject is a trauma or burn victim. 3.The method of claim 1, wherein said composition has a viscosity of 4 to20 centipoise at a temperature of 37 degrees Celsius using a cone andplate geometry with a cone angle of 0.02 radians and a constantfrequency of 30 rotations per minute.
 4. The method of claim 1, whereinsaid composition has a viscosity of 10 to 20 centipoise at a temperatureof 37 degrees Celsius using a cone and plate geometry with a cone angleof 0.02 radians and a constant frequency of 30 rotations per minute. 5.The method of claim 1, wherein said composition has a viscosity of 10 to15 centipoise at a temperature of 37 degrees Celsius using a cone andplate geometry with a cone angle of 0.02 radians and a constantfrequency of 30 rotations per minute.
 6. The method of claim 1, whereinsaid plasma expander consists essentially of: (a) from 0.1 to 5 percentby weight of said basic alpha keratose; (b) from 0 to 5 percent byweight of gamma keratose; and (c) from 90 to 99.9 percent by weight ofan electrolyte solution.
 7. The method of claim 1, wherein said plasmaexpander consists essentially of: (a) from 0.1 to 4 percent by weight ofsaid basic alpha keratose; (b) from 0 to 4 percent by weight of gammakeratose; and (c) from 92 to 99.9 percent by weight of an electrolytesolution.
 8. The method of claim 1, wherein when said plasma expander iscontacted to red blood cells forms aggregates of said blood cells ofless than 25 microns in diameter.
 9. The method of claim 1, wherein saidprocess further comprises the steps of precipitating said basic alphakeratose; re-dissolving said basic alpha keratose in a denaturingsolution, optionally in the presence of a chelating agent to complextrace metals; and re-precipitating said basic alpha keratose from saiddenaturing solution.
 10. The method of claim 9, wherein said denaturingsolution comprises a buffer solution.
 11. The method of claim 9, whereinsaid denaturing solution comprises a TRIS buffer solution.
 12. Themethod of claim 1 further comprising administering to said subject redblood cells (RBCs).
 13. The method of claim 12, wherein said red bloodcells are mammalian.
 14. The method of claim 12, wherein said red bloodcells are human.